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Biochemical and Pharmacokinetic Properties of <em>Aspergillus flavipes</em> Glutathione-Homocystine Transhydrogenase of Unique Affinity for Homocystine Reduction using GSH as Hydrogen Donor
ISSN: 2161-0444
Medicinal Chemistry

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Biochemical and Pharmacokinetic Properties of Aspergillus flavipes Glutathione-Homocystine Transhydrogenase of Unique Affinity for Homocystine Reduction using GSH as Hydrogen Donor

Ashraf SA El-Sayed1*, Abdallaa E Hassan2, Marwa A Yassin1, Hend MM Ibrahim3,4 and Asmaa M Hassan2

1Microbiology Department, Faculty of Science, Zagazig University, 44519, Egypt

2Chemistry and Biochemistry Department, Faculty of Science, Zagazig University, Egypt

3Proteomics and Metabolomics Facility, Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA

4Department of Medical Biochemistry, Faculty of Medicine, Zagazig University, 44519, Zagazig, Egypt

*Corresponding Author:
Ashraf S.A.El-Sayed
Microbiology Department
Faculty of Science, Zagazig
University, 44519, Egypt
Tel: 5856897330
E-mail: [email protected]; [email protected]

Received date: April 21, 2015; Accepted date: May 15, 2015; Published date: May 17, 2015

Citation: El-Sayed ASA, Hassan AE, Yassin MA, Ibrahim HMM, Hassan AM (2015) Biochemical and Pharmacokinetic Properties of Aspergillus flavipes Glutathione- Homocystine Transhydrogenase of Unique Affinity for Homocystine Reduction using GSH as Hydrogen Donor. Med chem 5:203-210. doi:10.4172/2161-0444.1000265

Copyright: © 2015 El-Sayed ASA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Glutathione-Homocystine Transhydrogenase (GHTHase) was characterized from Aspergillus flavipes as a novel enzymeof high specificity to reduce homocystine to homocysteine using GSH as hydrogen donor. GHTHase was further conjugated to mono-functional carboxyl polyethylene glycol (PEG) to improve its catalytic properties for various therapeutic uses especially against homocystinuria. The biochemical properties of free and PEG-GHTHase were assessed. The enzyme molecular mass was increased by 1.2 % (from 80 to 95 kDa) by PEG conjugation. The free and PEG-GHTHase have the same pH stability (6.5-8.0) and thermal stability (T1/2 1.0-1.3 h, at 50°C). Kinetically, the affinity and catalytic efficiency of PEG-GHTHase was decreased by 15% to GSH as hydrogen donor for reduction of homocystine than free enzyme. PEG-GHTHase has a slightly stability for suicide inhibitor as propargylglycine, hydroxylamine and iodoacetate. Upon proteolysis, the free enzyme retains less than 10% of its initial activity, comparing to 85.8 % of PEG-GHTHase upon trypsinand acid protease digestion for 30 min in vitro. From the pharmacokinetics properties in New Zealand rabbits, the half-life time of free and PEG-GHTHase was 10.0-12.2 h. Upon external infusion of NADH (20 μM) after 24 h of initial enzymes dosing, the half-life times of the enzymes were increased to 10 h. The biochemical and hematological parameters upon enzymes infusion were relatively not affected along the tested period. In vivo, GHTHase exhibits higher affinity to methionine and cysteine. Based on in vitro and in vivo biochemical properties, PEG-GHTHase could be a reliable enzyme in reduction of homocystine using GSH as electron donor. We assume, with more crystallographic studies, that GHTHase will be a new finding against various disorders-dependent homocystinuria.

Keywords

Aspergillus flavipes; Glutathione-Homocystine Transhydrogenase (GHTHase); PEG-GHTHase; Biochemical properties

Introduction

Glutathione-homocystine transhydrogenase (GHTHase, EC 1.8.4.1) is an enzyme catalyzing the oxidation/ reduction reactions of glutathione (GSH) and homocystine (Hcy2) to oxidized glutathione (GSSG) and homocysteine (Hcy) [1,2]. GHTHase is homodimeric protein of about 28 kDa and 60kDa with high catalytic kinetic and molecular structure of glutathione S-transferase [3].

Homocysteine is unnatural sulfur amino acid, intermediates in methionine cysteine cycle, easily oxidized in the blood to homocystine. Elevations in plasma homo-cysteine may be due to the genetic defects in its catabolizing enzymes or nutritional deficiencies in vitamin cofactors. Recently, homocysteinuria was endorsed as the main risk factor for various cardiovascular diseases such as atherosclerosis, thrombogenicity, mal endothelial function, atherothrombosis, Alzheimer's and diabetics [4-8]. The toxicity of homocystine arises from its strong chemical stability, insolubility, with feasibility of accumulation in small arteries causing atherosclerosis [7,8]. Thus, GHTHase could be a novel promising enzyme catalyzing the reduction of toxic insoluble homocystine to easily metabolized homocysteine using glutathione (GSH) as electron sources. Catalytically, GHTHase has a similar action of glutathione S transferase involved in detoxification/ oxidation of xenobiotic compounds using reduced glutathione (GSH) as hydrogen donor [9]. Thus, inhibition of human glutathione S transferase was emerged as promising anticancer strategy to overcome drug resistance [11] as GHTHase was firstly isolated and identified from human livers tissues [2]. So, it could be concluded that GHTHase might be implicated by indirect way with the action of glutathione S transferase. Further molecular and crystals structure of GHTHase will clearly elucidate this mystery.

However, from our previous catalytic and kinetic studies, GHTHase exhibit a relatively different pattern of catalysis even with subunit structural similarity with fungal GST [3]. Consequently, the physiological behavior of GHTHase in human blood, especially for removal of toxic homocystine, did worth to be elucidated. The yield of GHTHase from the fungal isolate was nutritionally optimized, as well as the enzyme was purified to its electrophoretic mobility using reliable chromatographic approach [3].

Thus, this work was extended to evaluate the in vivo pharmacokinetic properties of GHTHase, in addition to conjugate this enzyme with mono-functionalized carboxyl polyethyleneglycol (PEGylation) in order to increase its catalytic potency and structural stability for maximum exploitation to reduction of homocystine.

Since, PEGylation was one of the eminent processes for engineering of therapeutic enzyme [12] to increase their therapeutic and catalytic, as well as to diminish their antigenicity and protection against in vivo proteolytic attack [13-15], the objective of this context was to PEGylate the purified GHTHase, study the biochemical and catalytic properties of both enzymes. As well as to evaluate their stability in vitro against proteolytic attack, to assess the pharmacokinetic and antigenic properties of both enzymes in parallel using New Zealand Rabbits in vivo.

Materials and Methods

Animals

New Zealand Rabbits (6–8 weeks age) were purchased from vivarium of Faculty of Veterinary Medicine, Zagazig University, acclimated for 7 days in cages at room temperature, before injections.

Purification, PEGylation, Surface amino groups modification of A. flavipes GHTHase

GHTHase was purified from the cultures of AAspergillus flavipes as described [3]. The purified enzyme was conjugated to Methoxy polyethylene glycol propionic acid N-succinimidyl ester 5 kDa (PEG) by 1: 50 molar ratio according to [16] as illustrated in Scheme (Figure 1A and 2) The modification of terminal ɛ-amino groups on enzyme surface upon PEGylation reactions was estimated by fluorescamine assay [1,17]. The proposed reaction of fluorescamine reagent to enzyme surface lysine groups giving fluorophor with maximum fluorescence emission spectra at 450 nm at 290 nm excitation was illustrated in Fig. 1B. The surface primary free amino groups (glutamine, asparagine, arginine and lysine) were assessed by Ninhydrin reagent [18]. The enzyme surface free thiols was determined using DNTB (Elman's reagent) [19].

medicinal-chemistry-GHTHase-conjugated-Methoxy

Figure 1: PEGylation reaction of the purified A. flavipes GHTHase (A). GHTHase was conjugated to Methoxy Polyethyleneglycol Propionic acid N-succinimidyl ester (PEG) 5kDa in potassium phosphate buffer (100 mM, pH 7.8). The conjugation was conducted via peptide bond of enzyme surface lysine groups and carboxylic groups of PEG moieties. (B) Fluorescamine assay for reaction with surface lysine amino groups, the emitted fluorescence due to the fluorophor of enzyme-conjugated dye was measured at 450 nm.

medicinal-chemistry-modification-surface-functional

Figure 2: The modification of the surface functional groups of GHTHase upon PEGylation.

Activity of GHTHase

The activity of GHTHase was designed based in dual enzymatic reactions as described in our previous study [3]. The assay based on reduction of homocystine (Hcy2) by GHTHase to homocysteine (Hcy), which Hcy was subsequently γ-eliminated by homocysteine γ-lyase, forming α- ketobutyrate that detected by MBTH assay [20]. Briefly, the reaction contains 0.4 mM Hcy2, 20 mM GSH, 150 μl potassium phosphate buffer (100 mM) and 50 μl of enzyme preparation in 1 ml total volume. The reaction was incubated for 20 min at 40°C, then boiled for 10 min, centrifuged at 5000 rpm for 5 min. Blanks of enzyme and substrate were prepared at the same time. One ml of the supernatant was incubated with 100 μl of homocysteine γ-lyase [21] (1.7 U/ml) for 15 min at 40°C then the reaction was stopped by 100μl of TCA (10%) followed by 100 μl of MBTH reagent (0.025% MBTH dissolved in sodium acetate buffer pH 5.2). The mixture was incubated at 50°C for 30 min and the developed yellow color was measured at 330 nm. The activity of the GHTHase was calculated from the authentic curve of homocysteine (10-100 mM) using homocysteine γ-lyase (1.7 U/ml). The released α-ketobutyrate was quantified by MBTH assay [20].

Molecular weight determination of PEG-GHTHase

The proceeding of PEGylation reaction was evaluated from the size increasing by DLS analysis according to [22] with slight modifications. Briefly, the molecular size of free GHTHase and PEG-GHTHase was measured under the same conditions at the same dilution in potassium phosphate buffer using Zetasizer (Malvern, Nano-ZS, Malvern Instrument Limited, Grovewood Road, Malvern, UK). The molecular weights of the samples were calculated based on the size authentic molecularly known proteins. The authentic proteins were ovalbumin (44 kDa), canalbumin (75 kDa), aldolase (158 kDa) and ferritin (440 kDa).

Biochemical properties of the purified GHTHase and PEGGHTHase

The biochemical properties of free and PEG-GHTHase at optimum pH; pH stability, thermal kinetic properties and kinetics of substrate specificity were determined as described in the preliminary study on partial characterization of GHTHase [3].

The effect of various specific amino acid suicide analogues/ inhibitors on the activity of enzymes were assessed by pre-incubation with propargylglycine, iodoacetate, hydroxylamine (0.1-1.0 M) for 1 h, then measuring the residual activity under standard assay conditions.

Pharmacokinetic studies of free and PEG-GHTHase

The pharmacokinetic properties of the free and PEG-GHTHase were assessed using New Zealand white rabbits. Single dose (1 ml) of free GHTHase (59.33 U/mg per 1.5 ± 0.1 kg body weight) and PEGGHTHase (32.64 U/mg per 1.5 ± 0.1 kg body weight) was injected into rabbits that were acclimated for 50 days under controlled optimum physical conditions. Each treatment and control was a group of three rabbits. Primates plasma (zero time incubation) of injected and uninjected enzyme were used as positive and negative controls. The blood samples were collected intervally in EDTA containing tubes, centrifuged at 10,000 rpm for 2 min at 4°C, frozen at 0°C, till use. The activity of plasma GHTHase, plasma amino acid constitution as well as the biochemical and hematological parameters were assayed intervally as follows.

Activity assay of plasma GHTHase

The activity of free GHTHase and PEG-GHTHase in plasma was assayed as described above with slight modifications. Briefly, the mixture contains 50 μl of plasma, 0.4 mM homocystine, 20 mM GSH and 140 μl of 100 mM potassium phosphate buffer in 1 ml total reaction volume. The activity under standard assay as mentioned above.

Amino acid Constitution in vivo in response to free and PEGGHTHase

The effect of the formulated enzymes to the amino acids pool in vivo was traced by amino acid analyzer system (Amino acid analyzer LC3000 Eppendrof Germany) [23], regarding to negative and primate positive control.

Blood biochemistry of rabbits harboring free-GHTHase and PEG-GHTHase

To evaluate the biological effects of free GHTHase and PEGGHTHase on blood chemistry of treated rabbits, various hematological and biochemical parameters were determined [24,25]. The hematological parameters such as RBC, WBC, platelets, hemoglobin, hematocrit were analyzed (Erma INC automated hematology analyzer). Also, the biochemical parameters as ALT, AST, GGT, ALP, total protein, albumin, cholesterol, glucose, creatinine, LDH and triglycerides were assessed by automated chemistry analyzer (Dimension RXL Autoanalyzer). Positive and negative controls were considered at zero time for both treated and non-treated rabbits, respectively.

Effect of external infusion of NADH on reconstitution of GHTHase activity

The effect of external infusion of NADH on reconstitution of active GHTHase was evaluated according to [26] with slight modifications. After 12 h of the single dosing by free-GHTHase and PEG- GHTHase, the rabbits were i.v. injected by 1 ml of 10 mM NADH (0.11 mM final conc.). Blood sample were collected intervally every 5 h for 2 successive days, and the enzyme activity and spectral analysis were determined.

Statistical analysis

All the experiments were performed in triplicates. A student t- test was used to estimate the average and standard deviation.

Results

Purification and PEGylation of A. flavipes GHTHase

GHTHase was purified from A. flavipes as described previously [3] with specific activity (175.1 U/mg). The purified enzyme was PEGylated as described in Materials and Methods, the specific activity of PEGGHTHase was 106.2 U/mg, based on standard assay with relative activity 60.7% compared to free enzyme. The modification of enzyme surface functional groups was assessed based on the free ε-amino; primary amino groups and thiols. Based on the Ninhydrin assay, the ratio of free primary amino groups of PEG-GHTHase was 78.9% comparing to free enzyme, suggesting that about 21% was conjugated with PEG moieties. While, using Fluorescamine assay, the ratio of free ε-amino groups of PEG-GHTHase was about 47% to the non-PEGylated enzyme, thus, it could be deduced about 50% of the surface lysine was conjugated by peptide bond with PEG residues. However using DTNB assay, the free surface thiols of PEG-GHTHase was about 98.0% to the free enzyme, so, the little change on ratios of surface thiols, might be confirm the nil interaction of PEG residues with surface cysteine or methionine residues. The modification of the surface functional groups of GHTHase upon PEGylation was illustrated in Fig. 2.

Molecular size of free GHTHase and PEG-GHTHase

The molecular weight of the free and PEGylated enzyme was measured by DLS, comparing to authentic proteins as described in Materials and Methods. From the results (Figure 3A,B), size of the free enzyme the DLS was increased from 8.8 to 10 nm, thus, the molecular weight of the free GHTHase was predicted to be increased from 80 kDa to 95 kDa for PEG-GHTHase. This, results being reasonable with the degree of modification of surface amino groups, since, the free enzyme and PEG moieties was used in 1: 50 molar ratio.

medicinal-chemistry-Molecular-size-analysis

Figure 3: Molecular size of free GHTHase and PG-GHTHase by DLS analysis.

Biochemical properties of free GHTHase and PEG- GHTHase

The optimum reaction temperature for activity of free and PEG enzyme was estimated under standard assay using GSH and Hcy2 as substrates at different incubation temperatures (30, 37, 40, 45, 55, 65°C). From the obtained results the highest activity for both enzymes was measured at 37-40°C, with subsequent decreasing to the enzymes activities at higher temperature (Supplementary Figure 1). At 65°C, the activity of free enzyme was decreased by 55% comparing to less that 15% for PEG-GHTHase. The thermal stability of the enzymes was determined by pre-incubation without substrate at various temperatures (40, 50, 60°C), then measuring their residual activities under standard assay. From results (Data not shown), the free and PEG- GHTHase have the same response to thermal denaturation. At 50°C, both enzymes have a relatively similar half-life time (T1/2) from 1.0 to 1.3 h. However, the rate of thermal denaturation (Kr) for the free and PEG-GHTHase was 0.03 U/min. From the thermal kinetic parameters, PEGylation didn’t strongly stabilize the tertiary structure of the enzyme.

Also, the pH stability and pH precipitation of the free and PEGGHTHase were studied in different buffers ranged from 3.0 to 10.0. From the results (Data not shown), the free and PEG-GHTHase have the same range of pH stability from 6.5-8.0. At the lower (pH 3.5) and high (pH 10.0), the activity of both forms of GHTHase was decreased by 50 and 30 %. Thus, it could be suggested that PEGylation has no effect on the ionic state of GHTHase. From the pH precipitation profile, both enzymes were highly precipitated at pH rang 5.3 to 5.6, with no obvious precipitation at neutral to alkaline pH's.

Catalytic properties of free and PEG-GHTHase

The affinity of GHTHase towards various sulfur amino acids as hydrogen donors regarding to homocystine as standard hydrogen acceptor was evaluated based on the released α-ketobutyrate and ammonia as described in our previous preliminary study [3]. The free enzyme displayed a higher affinity to glutathione (100%) followed by lysine and cysteine as hydrogen donors for homocystine as an acceptor. Thus, the kinetics of substrate specificity of the PEG-GHTHase was determined based on Hcy2 as standard electron acceptor and GSH, cysteine and lysine as electron donors. From the results (Table 1) the PEG-GHTHase has a lower affinity to GSH (Km 20.1 mM) comparing to free-GHTHase (Km 14.3 mM), as well as lower Vmax value that were 277.7 and 212.7 U/mg/min, respectively. The turnover number of free GHTHase (Kcat 0.078 S-1) was reduced by about 26% compared to PEGGHTHase (Kcat 0.06 S-1) using GSH as hydrogen donor. The catalytic efficiency of the free GHTHase was slightly reduced for all tested electron donors compared to PEG-GHTHase under standard assay.

Substrate Free GHTHase PEG-GHTHase
Km (mM) Vmax
Umg-1 min-1
Kcat
min-1
Kcat/Km
mM-1 min-1
Km(mM) Vmax
Umg-1 min-1
Kcat
min-1
Kcat/ Km
mM-1 min-1
Glutathione 14.3 ± 2.0 277.7 ± 65 0.078 0.0063 20.1 ± 4.0 212.7 ± 57 0.06 0.0029
L-Cysteine 15.1 ± 2.3 200.1 ± 43 0.056 0.0004 17.2 ± 3.2 136.9 ± 29 0.038 0.0023
L-Lysine 40.2 ± 4.2 120.0 ± 25 0.043 0.0012 13.2 ± 2.9 133.3 ± 30 0.037 0.0029

Table 1: Kinetics of substrates specificity for free and PEG-GHTHase towards glutathione, L-cysteine and L-lysine as hydrogen donors acceptor. Means and SE for three replicates were calculated using student t-test.

Similar pattern for L-cysteine as hydrogen donor was observed, the affinity and reactivity of PEG GHTHase (Km 17.2 mM, Vmax 136.9 U/ mg/min) was decreased by 15 % compared to free GHTHase (Km 15.1 mM, Vmax 200 U/mg/min). Catalytically, L-lysine was used as electron donor by GHTHase for reduction of Hcy2. The slight oxidative activity of this enzyme was noticed collectively from its potency to oxidize cysteine and lysine. The affinity of free GHTHase to utilize GSH and L-cystine as hydrogen donor was quite similar, and about 2.8 time higher than L-lysine.

Effect of inhibitors on activity of free and PEG-GHTHase

The effect of suicide inhibitors targeting specific amino acid was used to elucidate the chemical identity of GHTHase as described in our preliminary study [3]. Among the selected inhibitors, propargylglycine (PPG); hydroxylamine (HA) and iodoacetate (IA) as powerful GHTHase inhibitors were selected to verify the protective effect of PEG moieties. The kinetic inhibitory parameters for both enzyme forms were summarized in Table 2. Practically, a relative higher resistance exerted by PEG-GHTHase than free enzyme to the tested inhibitors was revealed from the IC50 values, indicating the slight protection of enzyme structure by PEG residues. The IC50 value for PPG, HA, IA was 3.3, 2.2, 1.4 mM for free GHTHase while for PEG-GHTHase was 3.8, 2.7 and 1.6 mM, respectively. The mode of inhibition of the compounds was evaluated using the BOTDB software (http://www.botdb.com). The highest Ki values were detected for the noncompetitive mode for PPG followed by HA and IA for PEG-GHTHase, then free GHTHase, respectively. The noncompetitive Ki for PPG was 3807 and 3266 μM, for HA was 2675 μM and for IA was 1580 and 1362 μM for PEGGHTHase and free enzyme, respectively.

Inhibitor   Inhibition mode Free-GHTHase PEG-GHTHase
  Propargylglycine IC50 (mM)   3.3 3.8
  Ki (µM) Competitive 726 1091
Noncompetitive 3266 3807
Uncompetitive 2540 2715
Hydroxylamine IC50 (mM)   2.2 2.7
Ki (µM) Competitive 489 767
Noncompetitive 2199 2675
Uncompetitive 1710 1908
Iodoacetate IC50 (mM)   1.4 1.6
Ki (µM) Competitive 303 453
Noncompetitive 1362 1580
Uncompetitive 1059 1126

Table 2: Kinetic parameters of inhibition of free and PEG-GHTHase for specific suicide inhibitor.

In vitro proteolysis of free and PEG-GHTHase

Enzyme proteolysis was assessed in vitro by incubation of the enzymes with acid protease and trypsin (10 U/ml) for 30 min at 37°C. The residual enzymatic activity was evaluated by colorimetric standard assay and by SDS-PAGE. From the obtained data (Figure 4), the activity of free GHTHase was strongly reduced by 90 % upon proteolysis by the two proteases after 15 min. In contrary, the PEG-GHTHase retains 85.8 % and 76.8% of its activity upon digestion by trypsin for 15 and 30 min, respectively. Also, in case of using protease K, the PEG-GHTHase retains 55.1 and 44.8 % of its initial activities after 15 and 30 min, respectively. Thus, it could be concluded, PEGylation of GHTHase relatively masks the recognition sites for proteases.

medicinal-chemistry-Relative-activity-proteolytic

Figure 4: Relative activity of free and PEG-GHTHase upon proteolytic cleavage, using Trypsin and protease K. The enzyme was incubated with 10 U/mg of each protease for 30 min, the residual activity of GHTHase was determined under standard assay as descried in Materials and Methods.

Pharmacokinetic properties of free and PEG-GHTHase in New Zealand rabbits

The pharmacokinetic properties of the enzymes were determined using single intravenously dose of free (175.1 U/mg) and PEG-GHTHase (106.2 U/mg) to the rabbits (1.5 ± 0.3 kg). The pharmacokinetic parameters were summarized in Table 3. Apparently, the biological half-life time of holo-PEG-GHTHase was 12.2 h comparing to free GHTHase (10 h), while the elimination constant was 0.02 U/h for both apo-enzymes. After 24 h of enzyme circulation, 20 μM of NADH as predicted co-enzyme was intravenously injected to the rabbits and the activity of the enzymes were determined by the standard assay. The half-life time of the free and PEG-GHTHase was increased to 9.6 and 12 h, respectively. This increment on the enzyme activities, might be slightly assumes the NADH dependence as coenzyme in vivo. The relative prolongation in half-life time for PEG-GHTHase than free one, assumes the masking effect of surface GHTHase antigenic and proteolytic sites by PEG moieties.

Kinetic parameters Free-GHTHase PEG-GHTHase
Enzyme injection  NADH Injection Enzyme injection  NADH Injection
Initial Drug Conc. (U/ml)* 0.698 - 0.384 -
Biological half-life time (h)** 10 9.6 12.2 12
Elimination Constant (U/h)*** 0.016 0.016 0.010 0.016

Table 3: in vivo Pharmacokinetic properties of free and PEG-GHTHase using New Zealand Rabbits.

To verify the enzymes affinity to amino acids in vivo, the concentration of serum amino acids in rabbits in response to injection by free and PEG GHTHase was determined by amino acid analyzer. From the profile of amino acids (Table 4), an obvious fluctuation on the concentrations of serum amino acids in response to free and PEGGHTHase injection was detected compared to negative control (noninjected). However, the free and PEG-GHTHase displayed a higher affinity to hydrolyze L-methionine, cysteine and lysine, compared to other amino acids. The concentration of L-methionine and cysteine was reduced by about 11 and 5 fold for the free GHTHase and by 7.5 and 3 fold for PEG-GHTHase, respectively, after 6 h of injection compared to non-enzymes injected controls. The concentration of serum L-arginine and lysine was approximately reduced to its half initial titers, after 6 h of injection by free and PEG-GHTHase. Apparently, the titer of aspartic acid, threonine, valine, isoleucine, tyrosine and proline were not strongly affected by the injection of free and PEG-GHTHase comparing to negative control. Otherwise, after 6 h, the titer of serum amino acids as aspartic acid, serine, glycine, alanine and histidine were noticeably increased over the control, upon free and PEG-GHTHase injection. Practically, the higher affinity of GHTHase in vivo towards cysteine and lysine was greatly reliable with the amino acid analysis.

Amino acid Negative control (µM) Free-GHTHase (µM) PEG-GHTHase (µM)
Aspartic acid 181.36 177.6 147.04
Threonine 85.40 68.96 75.12
Serine 146.17 290.32 716.4
Glutamic acid 647.17 936.96 743.48
Glycine 108.68 398.48 155.96
Alanine 123.08 156.08 140.0
Valine 132.53 132.96 104.8
Methionine 330.68 25.9 47.6
Cysteine 267.0 57.9 89.0
Isoleucine 129.06 165.8 149.6
Leucine 266.86 261.36 865.04
Tyrosine 500.0 559.04 508.88
Phenylalanine 45.58 84.8 107.76
Histidine 120.38 110.0 138.4
Lysine 212.48 105.2 122.88
Arginine 266.03 116.08 125.0
Proline 378.935 348.5687 369.9019

Table 4: Serum amino acid concentration of rabbits in response to injection of free and PEG-GHTHase after 6 h compared to negative control.

Biochemical and hematological parameters of rabbits in vivo in response to free and PEG-GHTHase

The biochemical and hematological responses upon injection of the free and PEG-GHTHase were determined after 2 and 6 h and compared to negative controls (Table 5). From the obtained data, the titer of blood urea, ALT and total protein was slightly reduced upon using PEGGHTHase comparing to free GHTHase along the circulation time, with slightly no effect on the levels of blood creatinine, albumin and blood AST. However, the activity of ALP was increased by 5-7 times in response to both forms of GHTHase, in contrary to the significant reduction of platelets titer by two times, compared to negative controls. Otherwise, the concentration of RBCs and human globulins was not affected by the enzyme injection. From these results, the enzymes have no negative effect on various tested biochemical and hematological parameters, except the induction of alkaline phosphatase.

  Test   Free-GHTHase PEG-GHTHase
Negative Control 2 h 6 h 2 h 6 h
Urea (mg/dl) 46.1 ± 8.1 43.6 ± 2.9 47.1 ± 2.68 42.5±17.2 42 ± 16.2
Creatinine (mg/dl) 0.9 ± 0.1 0.77 ± 0.2 1.05 ± 0.07 0.87± 0.06 0.9  ± 0.06
ALT (U/L) 46.1 ± 4.4 39.5 ± 4.1 35.4 ± 2.26 28.8±6.7 35.7 ±  2.4
AST (U/L) 35.5 ± 2.7 35.7 ± 3.0 33.5 ± 3.53 33.3±3.35 32.3 ± 2.7
Albumin (g/dl) 3.1 ± 0.1 2.8 ± 0.2 2.95 ± 0.07 3.1±0.17 3.1 ±  0.115
Total protein (g/dl) 7.1 ± 0.3 5.5 ± 0.2 5.85 ± 0.07 5.7±0.15 6.1 ± 0.15
ALP (U/L) 31.1 ± 5.7 206.7 ± 13 182.5 ± 9.2 173.3  17 197.6 ± 10.8
PLT (X 109/L) 435 ± 51 220.3 ± 14 190.5 ± 16 210.3± 19 186.3 ± 15.6
WBCs (X 109/L) 10.2 ± 2.3 8.6 ± 3.2 7.4 ± 0.1 6.86   0.7 5.4 ± 0.6
RBCs (X 1012/L) 4.52 ± 0.2 5.2 ± 0.1 5.2 ± 0.09 6 0.15 5.2 ± 0.4
HGB (g/dl) 10.53±0.1 10.7 ± 0.35 10.2 ± 0.2 10.6 0.25 8.4   3.2

Table 5: in vivo Biochemical and hematological Parameters of rabbits in response to free-GHTHase.

Discussion

The present study was focused on biochemical characterization of GHTHase based on its specificity for reduction of homocystine (Hcy2) using reduced glutathione (GSH) as hydrogen donor. Oxidation of soluble homocysteine to highly insoluble homocystine is the main noticeable biochemical reason raised for various cardiovascular diseases, atherosclerosis, Alzheimer and diabetes [5,8]. GHTHase was characterized in our preliminary studies [3] from AAspergillus flavipes, exhibiting a unique catalytic and biochemical properties comparing to glutathione-s-transferase. Thus, this study was motivated to positively modify the GHTHase via polyethylene glycol conjugation and assess the catalytic and structural properties of PEG-GHTHase comparing to unmodified enzyme, in vitro and in vivo. Biochemical characterization of this enzyme, could be lay the core for exploitation against elevated Hcy2 cardiovascular diseases.

The purified A. flavipes GHTHase was PEGylated, under standard conditions reported by [1,16]. The relative activity of PEG-GHTHase was 60.7 % regarding to unmodified GHTHase. The reduction of the enzyme activity upon modification might due to the direct interaction with the catalytic sites causing slight hindrance to binding of substrates, as reported for modified enzymes by PEGylation [1,3,16,26,27]. Practically, the colorimetric activity was correlated with the modification of surface primary and ε-amino groups of lysine residues, ensuring the little interaction with surface amino groups [27]. However, there is no notable modification of free surface thiols upon modification by PEG moieties, that could by justified by the strict selectivity for amino groups modification or absence of surface thiols. Further molecular and crystallographic analyses, were undergoing to figure out the enzyme surface functional groups. The extent of PEGylation reaction was verified from the increment of GHTHase molecular size, by SDSPAGE and DLS analysis. For the native-PAGE, the two forms display the same mobility with slight broadness to band of PEG-GHTHase, that might to the polydispersivity of PEG moieties, while, the similar mobility, might be to lower molar ratio of PEG moieties to enzyme (50/1) [1]. However, the distinct increasing on the molecular weight of METase by PEGylation [16], might be to the higher used PEG ratio (120/1). Using the DLS, the increasing of molecular size was clearly observed from 80 to 90 kDa, that being more reliable to assess the extent of modification, than native-PAGE. From the physicochemical properties, the free and PEG-GHTHase have the same response to pH stability and thermal stability, this imply, modification by PEG moieties had no effect on structural identity of the enzyme. The catalytic affinity of the free and PEG-GHTHase was assessed towards homocystine as hydrogen acceptor and various hydrogen donors. The affinity of PEGGHTHase for glutathione as standard hydrogen donor was decreased by 30 % than free GHTHase, that consistent with ratio of reduction of activity of various PEGylated enzymes as reviewed by [12,28].

To verify the mode of conjugation of PEG moieties to GHTHase, various specific inhibitors for cysteine, lysine and glutamic acid, as PPG, HA and IA, respectively, was used (Table 2). PEG-GHTHase displays fairly resistance to PPG, HA and IA, comparing to native enzyme, suggesting the slight protection by PEG moieties to the surface enzyme amino acids. Practically, biochemical characterization using specific substrates analogues/ inhibitor to explore the modification of enzyme surface amino acids was frequently documented [1,29-32].

The protective role of PEG moieties on GHTHase from proteolysis by acid protease and trypsin was assessed. From the colorimetric activity and molecular homogeneity (by SDS-PAGE), the PEGGHTHase displays higher resistance to proteolysis towards the two proteases than free GHTHase. Upon trypsinolysis, the PEG-GHTHase retains more than 85% of its initial activity comparing to less than 10 % to free GHTHase. Masking of the surface recognition proteolytic sites by PEGylation was one of the main practical affordable benefits, to increasing the enzymes half-life time by decreasing their proteolysis, in vivo [1,26,33]. Higher induction of trypsin levels in blood plasma is a noticeable biochemical response to almost of enzymes dependent therapies [34]. Practically, trypsin usually attacks the peptide bonds at recognition sites Lys53- Gly54, and Arg206-Ser207 [33]. Since, we utilized modified PEG residues, specifically to makes conjugates with surface lysine, thus the extending of enzyme activity and relative structural stability against could be justified to the inaccessibility to lysine residues to cleavage by trypsin [35]. Lowering the accessibility of proteolytic recognition sites by PEGylation was extensively reviewed [28].

From the pharmacokinetic parameters, the half-life time of PEGGHTHase, was slightly increased by about 1.3 fold comparing to free one. By infusion of NADH (20 μM), the activity of both enzyme forms was duplicated, suggesting the involvement of NADH as co-enzyme. The NADH dependence of this enzyme was reported by [2]. For the scarce documents on the catalytic and structural identity of GHTHase, so, further molecular and crystallographic studies were undergoing to elucidate its catalytic and co-enzyme identity. To verify the enzymes affinity in vivo, the concentration of serum amino acids in rabbits in response to injection by free and PEG GHTHase was determined by amino acid analyzer. GHTHase displayed a higher affinity to hydrolyze L-methionine, cysteine and lysine, comparing to other amino acids, in vivo. L-Methionine and cysteine titers were reduced by about 11 and 5 fold in response to free GHTHase and by 7.5 and 3 fold for PEG-GHTHase, respectively, after 6 h of injection, comparing to nonenzymes injected controls. However, homocystine was not detected in blood serum by HPLC for healthy animals. The higher affinity of GHTHase in vivo towards cysteine and lysine was greatly comparable with the in vivo amino acid analysis. From the biochemical parameters, the titer of blood urea, ALT and total protein was slightly reduced upon using PEG-GHTHase comparing to free GHTHase along the circulation time, with no effect on the levels of creatinine, albumin and blood AST. However, the activity of ALP was increased by 5-7 times in response to both forms of GHTHase, in contrary to the significant reduction of platelets titer by two times, comparing to negative controls. Similar biochemical responses were reported for various therapeutic enzymes [26,36].

It worth mentioning that, the present findings suggest the presence Glutathione S-Transferase (GST) like enzyme with unique specificity to reduce Hcy2 to Hcy using reduced glutathione GSH as hydrogen donor, designated as Glutathione Homocystine Trans-hydrogeanse (GHTHase). This enzyme was firstly isolated from human liver [2], and this is first report to characterize this enzyme from microbes. To date, the information about fungal GST is limited, only few classes were studied, and the property of each class was dependent on the fungal isolate producing it [37]. However, the distribution of different forms of GSTs, could be to maintains the cellular redox state by sequestering the ROS [38]. Thus, we suggest characterization of GST Like GHTHase from fungi will clearly explores different modes for fungal oxidation of xenobiotic using GSH, from view of fungal proteomics. Additionally, GHTHase with high specificity to reduce Hcy2 based on GSH as hydrogen donor could be used as mediator to organosulfur prodrugs as isothiocyanates for anticancer therapy [39]. Currently, the versatility of GST activity for motivation conversion of various prodrugs, to active anticancer agents was received much insights [40,41]. Moreover, GHTHase with high specificity to eradicate toxic compounds (Hcy2) as well as prodrugs mediation will shed the light to its powerful combinatorial uses in various therapies.

In conclusion, GHTHase of unique activity to reduce homocystine using GSH (H2 donor) was purified from AAspergillus flavipes, with relative biochemical obvious similarity to glutathione S-transferase. PEGylation of GHTHase was tried to improve the enzyme biochemical properties. PEG-GHTHase displayed a plausible resistance to in vitro proteolysis upon using trypsin and acid protease. The enzymes have no observed cytotoxicity in vivo as reported from the biochemical and hematological properties of rabbits.

Acknowledgment

We greatly appreciate the funding support from Zagazig University, Egypt, to ASEA. We gratefully thank Prof. Sadik Esener, Nanoengineering Dep., University of California, San Diego, CA, USA, for his fruitful discussion. We also thank professor Salah E. Abdel-Ghany, Department of Plant Molecular Biology at Colorado State University, Fort Collins, CO, USA for his critical editing the manuscript.

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